Abstract

Interleukin 1α (IL-1α) is an important regulatory cytokine, the release of which after an injury can induce activation of transcription factors nuclear factor (NF)κB and activator protein (AP-1), which promote expression of genes involved in cell survival, proliferation, and angiogenesis. IL-1α is expressed autonomously by head and neck squamous cell carcinomas (HNSCCs) and a variety of other cancers, raising the possibility that IL-1α may serve as an autocrine factor that stimulates the activation of prosurvival transcription factors and target genes in cancer. In this study, we examined the role of IL-1α in the activation of NFκB and AP-1, the expression of proangiogenic cytokine IL-8, and in the survival and proliferation of HNSCC cell lines. HNSCCs were found to secrete and respond to functional IL-1α, in that culture supernatant from a high IL-1α-secreting line, UM-SCC-11B, could induce secretion of cytokine IL-8 by a low IL-1α-secreting line, UM-SCC-9; and the induction of IL-8 secretion could be blocked by the anti-IL-1α-neutralizing antibody or the IL-1 receptor antagonist (IL-1RA). Furthermore, IL-1α could induce the expression of IL-8 through an autocrine mechanism, in that transfection of UM-SCC-9 cells with a plasmid encoding IL-1α resulted in the increased coexpression of IL-1α and IL-8; whereas transfection with a plasmid encoding IL-1RA lacking the secretory leader sequence led to the decreased coexpression of IL-1α and IL-8. IL-1α was found to induce coexpression of IL-8 through the activation of NFκB and AP-1, in that mutation of the NFκB site within the IL-8 promoter abolished autocrine- and recombinant IL-1α-induced IL-8 reporter gene activity, whereas mutation in AP-1 partially decreased IL-8 reporter gene activity in UM-SCC-9 cells. Intracellular expression of IL-1RA decreased NFκB reporter gene activity, indicating that endogenously expressed IL-1α contributes to constitutive NFκB activation in this HNSCC line. Expression of IL-1α affected survival of UM-SCC-9, inasmuch as transfection of cells with plasmid encoding IL-1α or IL-1RA led to the increased or decreased survival of cells cotransfected with a β-galactosidase reporter gene, respectively. IL-1α was also found to promote the increased growth of UM-SCC-9 cells in vitro. We demonstrate that exogenous and endogenous IL-1α contributes to the transcriptional activation of NFκB and AP-1, to the expression of IL-8, and to cell survival and the growth of HNSCC in vitro.

INTRODUCTION

IL-15
is a cytokine released after stress or injury that mediates a variety of protective responses by the cell and the organism (1)
. IL-1 can induce the activation of immediate-early transcription factors and genes that promote the survival and proliferation of cells (2)
and cytokines that mediate protective inflammatory and angiogenesis responses (3, 4)
. Previously, we reported that HNSCCs constitutively express IL-1α and a repertoire of IL-1-inducible cytokines, including IL-6, IL-8, and GM-CSF (5)
. These cytokines have been detected in the supernatants of cell lines, in tumor specimens, and in serum from patients with HNSCC (5, 6)
. Several of these cytokines seem to contribute to the malignant phenotype of SCCs and other cancers (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
. IL-1α has been reported to directly enhance the proliferation of transformed human keratinocytes and cervical SCC cell lines (10)
. IL-8 has been shown to act as an autocrine growth factor in HNSCC, melanoma, and bronchogenic carcinoma, and IL-8 and GM-CSF have been reported to have proinflammatory and angiogenic activities that enhance tumor growth and metastatic potential (14, 15, 16, 17)
. However, the nature and biological importance of the mechanism(s) that initiate the coactivation of this repertoire of cytokines in HNSCC and other cancers has not been well defined.

In considering the possible mechanisms responsible for coexpression of IL-1α, IL-6, IL-8, and GM-CSF, we noted that the promoter region of all four cytokine genes share recognition sites for immediate-early transcription factors NFκB/Rel A and AP-1, and found that differences in cytokine expression correlated with activation of these transcription factors (18, 19, 20)
. Additional studies to define the contribution of NFκB/Rel A and AP-1 in the activation of the IL-8 gene in HNSCC demonstrated that mutation of the NFκB and AP-1 site within the IL-8 promoter reduced IL-8 reporter gene activity (18)
. The expression of a mutant inhibitor-κB in human or murine SCC cells inhibited the activation of NFκB and the expression of cytokines (19, 20)
. Furthermore, inactivation of NFκB in HNSCC inhibited cell survival and growth in vivo, indicating that the activation of NFκB plays an important role in the malignant phenotype of HNSCC (19)
. Activation of NFκB has also been detected and shown to prevent apoptosis in lymphomas and breast and prostate cancer (21)
, but the factor(s) responsible for the activation of NFκB in HNSCC and these other cancers has not been defined. Because IL-1 has been shown to be an inducer of NFκB activation and IL-8 expression (2, 3, 4)
, we hypothesized that the expression of IL-1α by HNSCC could play an important role in the autocrine activation of transcription factors NFκB and AP-1, the expression of cytokines such as IL-8, and the promotion of cell survival and growth.

Previously we explored whether IL-1α could promote the coexpression of IL-8 and GM-CSF in HNSCC, and we found that increased concentrations of recombinant IL-1α or the expression of IL-1α after transfection was able to induce IL-8 and GM-CSF production by HNSCC cell lines (5)
. In contrast, the addition of anti-IL-1α neutralizing antibody and IL-1RA blocked the cytokine production induced by exogenous IL-1α but had little effect on constitutive expression (5)
. In this study, we examined the effects of modulating exogenous and endogenously expressed IL-1α on the activation of NFκB, AP-1, and the expression of the proangiogenic cytokine IL-8; and on the survival and proliferation of HNSCC cell lines. We demonstrated that exogenous and endogenous IL-1α contributes to the transcriptional activation of NFκB and AP-1, to the expression of IL-8, and to the cell survival and growth of HNSCC in vitro.

MATERIALS AND METHODS

Antibodies and Plasmids.

Goat antihuman IL-1α, goat IgG control, and recombinant IL-1RA were purchased from R&D Systems, (Minneapolis, MN). Plasmid pIgk-luc (immunoglobulin κB-luc containing two copies of the NFκB binding site upstream of the minimal promoter fused with the luciferase gene) was described previously (22)
. IL-8 promoter reporter gene plasmids, in which expression of a luciferase gene is directed by a wild-type or a mutant IL-8 promoter, were described previously (23)
. These plasmids contain the 133-bp sequence upstream from the transcription start site of the IL-8 gene. Constructs were used which contain point mutations in the putative NFκB and AP-1 sites (23)
. PCMVLacZ consists of a LacZ gene inserted between the CMV promoter and bovine growth hormone poly(A) signal sequence in pcDNA3 (Invitrogen, Carlsbad, CA).

Construction of Vectors Encoding Sequence of IL-1α Sense, and IL-1RA.

To make vectors that express human IL-1α under control of a CMV promoter, a BamHI DNA fragment containing the complete coding sequence of human IL-1α was isolated from phIL1AcDNA (ATCC no. 65259) and ligated into pcDNA3 (Invitrogen) that was predigested by BamHI. Recombinants were screened for clones with inserts in sense orientation, yielding pcDNAhIL-1α (IL-1α). The construct lacking a signal sequence for secretion-encoding intracellular expression of IL-1RA (pcDNAicIL1-RA) was a kind gift from the laboratory of Dr. Steve Haskill, University of North Carolina, Chapel Hill, NC (24)
.

Culture of UM-SCC Cell Lines and Preparation of Supernatants for ELISA.

UM-SCC-9 and -11B lines were derived from human SCC of the upper aerodigestive tract, with informed consent, at the University of Michigan (Ann Arbor, MI). The cell lines were from patients exhibiting an aggressive clinical course who developed local or regional recurrence, and who died within two years of diagnosis (5, 6)
. The cell lines established from single isolates of a patient specimen are designated by a numeric designation; where established from isolates from two time points, the designation includes an alphabetical suffix, i.e., “A” or “B.” The cell lines were maintained in Eagle’s minimal essential media supplemented with 10% fetal bovine serum and penicillin/streptomycin. For ELISA, 12 ml of fresh medium were added to tumor cell lines when 60–80% confluent in 75-cm2 tissue culture flasks, and cells were collected after 48 h. Supernatants were centrifuged at 1500 × g to remove particles, and aliquots were stored frozen at −80° until use in ELISA. Cell number in the culture flasks was determined to standardize the quantity of cytokine secreted per 106 tumor cells.

Quantitation of Cytokine Production in Supernatants from SCC by ELISA.

ELISA kits for specific cytokines were purchased from R&D Systems and Endogen (Cambridge, MA) and used according to the manufacturer’s protocol. Each sample was tested in duplicate in each experiment. After development of the colorimetric reaction, the absorbance (OD) at 450 nm was quantitated by an 8-channel spectrophotometer (Bio-Tek Systems, Winooski, VT), and the OD readings were converted to pg/ml of cytokines based on standard curves analyzed in each assay. If the OD readings exceeded the linear range of the standard curves, the ELISA assay was repeated using serial dilutions of the supernatants. Each sample was tested by at least two independent ELISA measurements, and the data were calculated from the mean of the two to four tests for each sample. The lower limit of sensitivity of these ELISAs for detecting IL-1α and IL-8 was found to be 4 and 10 pg/ml, respectively.

Cell Extracts.

Cell extracts were made according to the method of Beg et al.(25)
, using a procedure from the laboratory of Dr. U. Siebenlist (National Institute of Allergy and Infectious Diseases, NIH) with minor modifications. Briefly, 1 × 106 cells were rinsed with PBS and harvested from tissue culture flasks by gentle scrapping. After spinning down the cells and removing the PBS, an equivalent volume of lysis buffer [50 mm Tris (pH 7.4), 100 mm NaCl, 50 mm NaF, 30 mm sodium PPi, and 0.5% NP40) was added to resuspend the cell pellet. A protease inhibitor cocktail tablet (Complete, Mini; Boehringer, Mannheim, Germany) was added per 10 ml of lysis buffer before use. Then samples were spun at 18,000 × g for 20 min at 4°C. Supernatants were aliquoted, snap frozen, and stored at −80°C. Protein concentrations were determined using a BCA protein assay kit following the manufacturer’s instructions (Pierce, Rockford, Illinois).

Cell Transfection and Reporter Gene Assays.

UM-SCC-9 and -11B cells were plated at 2 × 105 cells/well in a six-well plate and allowed to adhere overnight. For transfections, 2 μg of −133 IL-8-luc or pIgk-luc reporter DNA were added in 100 μl of OptiMEM medium, and mixed with 5 μl of Lipofectin (Life Technologies, Inc., Gaithersburg, MD) in 100 μl OptiMEM medium. pCMVLacZ or pRSVLacZ (0.2 μg) was cotransfected to confirm transfection efficiency. The mixture was incubated for 30 min, and 0.8 ml of OptiMEM was added to the DNA/Lipofectin solution and gently mixed. Then 1 ml of the solution was added to each well and the plates were incubated at 37° for 5 h. Then the transfection media was replaced with Eagle’s minimal essential medium plus 10% serum, and incubation of the cells was continued for 48 h. The cells were harvested and reporter gene activities were assayed using the Dual-Light Luciferase and β-Galactosidase Reporter Gene Assay System (Tropix, Bedford, MA), and chemiluminescence was measured by a Monolight 2010 luminometer (Analytical Luminescence Lab, San Diego, CA).

Measurement of Cell Proliferation by MTT Assay.

UM-SCC-9 or -11B cells (5 × 103) were plated in a 96-well microtiter plate and incubated overnight. Then the cells were washed twice with PBS and exposed to 250 pg/ml of IL-1α in serum-free KGM. Cell density was determined using an MTT cell proliferation assay (Boehringer Mannheim, Indianapolis, IN). MTT-labeling reagent was added at days 1, 3, and 5 after IL-1α treatment, and colorimetric ODs were measured at 570 nm by a microplate reader (Bio-Tek Systems).

RESULTS

IL-1α of HNSCC and Recombinant Origin can Induce IL-8 Secretion by UM-SCC-9 Cells.

Previously we showed that differences in IL-1α expression were associated with corresponding differences in IL-8 expression among HNSCC cell lines, and that the addition of recombinant IL-1α was able to stimulate IL-8 production by a low cytokine-producer cell line, UM-SCC-9 (5)
. To confirm whether IL-1α of HNSCC origin is functional and can induce IL-8 secretion by UM-SCC-9, we compared the abilities of recombinant IL-1α and an IL-1α-containing supernatant from the UM-SCC-11B cell line to induce IL-8 secretion (Fig. 1)⇓
. Although UM-SCC-9 cells can produce 30–50 pg IL-1α/106 cells over 5 days (19)
, the amount of IL-1α in control cultures of UM-SCC-9 cells by 24 h was below the 4-pg/ml threshold for detection by ELISA, as shown in Fig. 1A⇓
. IL-1α was detected after the addition of 500 pg/ml of rIL-1α, rIL-1α-plus-control antibody, or rIL-1α-plus-IL-1RA, but IL-1α was completely neutralized when rIL-1α-plus-anti-IL-1 antibody was added. Fig. 1B⇓
shows that, with low basal IL-1α expression, UM-SCC-9 produces a relatively low amount of IL-8 (106 pg/ml), when compared with the 1200 pg/ml of IL-8 detected after the addition of 500 pg/ml of recombinant IL-1α (Fig. 1B)⇓
. IL-1α-induced IL-8 production was completely inhibited by anti-IL-1 antibody or IL-1RA (Fig. 1B)⇓
. Anti-IL-1 antibody and IL-1RA did not effectively block the constitutive production of IL-8 by UM-SCC-9 cells. Next we tested a 48-h-conditioned medium from UM-SCC-11B cells, which contained ∼500 pg/ml of IL-1α by ELISA. In a pilot experiment, we tested IL-1α stimulatory activity for IL-8 induction in 100- or 50%-conditioned supernatant and found that 100%-conditioned supernatant showed stronger inducibility of IL-8 (data not shown). As shown in Fig. 1C⇓
, 24 h after the addition of UM-SCC-11B supernatant to UM-SCC-9 cell cultures, we were able to detect ∼300 pg/ml IL-1α, which was completely neutralized when anti-IL-1 antibody was added. Fig. 1D⇓
shows that UM-SCC-11B-conditioned medium (initially containing ∼500 pg/ml of IL-1α) induced a level of IL-8 production that is ten times higher (>12,000 pg/ml after subtraction of the IL-8 in UM-SCC-11B supernatant alone) than that induced (1,200 pg/ml) by an equivalent amount of rIL-1α. Fig. 1D⇓
shows that neutralization by anti-IL-1 antibody and IL-1R antagonist, but not by the control antibody, results in complete inhibition of inducible IL-8 production, indicating that IL-1α in UM-SCC-11B cell-conditioned supernatant is functional and can induce IL-8 production by UM-SCC-9 cells. These results suggest that, once IL-1α induces IL-8 expression, another factor(s) in the conditioned supernatants of UM-SCC-11B may enhance further the expression of IL-8 by UM-SCC-9 cells.

rIL-1α and UM-SCC-11B-conditioned medium induce IL-8 production by UM-SCC-9 cells. Conditioned medium from UM-SCC-11B cells was collected after 48 h from near-confluent cell cultures. UM-SCC-9 cells were plated at 5 × 104 cells/well in 24-well plates overnight, and 500 pg/ml rIL-1α in 1 ml of fresh medium or 1 ml of UM-SCC-11B-conditioned medium was added to each well of cultured UM-SCC-9 cells for 24 h. For neutralization experiments, anti-IL-1α antibody, isotype control antibody (10ng/ml), or IL-1RA was added to rIL-1α or UM-SCC-11B medium for 1 h before addition into the UM-SCC-9 cell cultures. After a 24-h incubation, the UM-SCC-9 supernatants were collected, and cytokines were measured by ELISA. A and B, quantitation of IL-1α and IL-8, respectively, in UM-SCC-9 culture supernatant after incubation with exogenous rIL-1α, anti-IL-1 antibody, and IL-1RA for 24 h. C and D, quantitation of IL-1α and IL-8, respectively, in UM-SCC-9 supernatant after treatment with UM-SCC-11B-conditioned medium for 24 h. D, IL-8 secretion by UM-SCC-9 after subtraction of the baseline level of IL-8 present in the UM-SCC-11B-conditioned medium. Supernatant from UM-SCC-11B cell cultures was collected and tested from two independent cultures and the data from one of three independent experiments is shown. ∗, a significant difference when compared with control (t test; P < 0.05).

We examined whether IL-8 production by UM-SCC-9 cells could be modulated by increases or decreases in endogenous IL-1α production. UM-SCC-9 cells were transiently transfected with plasmid DNA encoding the gene sequence for IL-1α and an icIL-1RA construct that is expressed intracellularly, and the cell culture supernatants were collected 48 h posttransfection. IL-1α-transfected UM-SCC-9 cells exhibited increased IL-1α (Fig. 2A)⇓
and IL-8 (Fig. 2B)⇓
production in supernatants by ELISA. In contrast, expression of an icIL-1RA construct that is expressed intracellularly resulted in significant inhibition of IL-1α (Fig. 2A)⇓
and IL-8 (Fig. 2B)⇓
expression. These results indicate that the expression of IL-1α by UM-SCC-9 cells can regulate the expression of IL-8 through an autocrine mechanism.

Regulation of IL-8 production by UM-SCC-9 after transient transfection of IL-1α or IL-1RA DNA constructs. UM-SCC-9 cells were plated at 5 × 104 cells/well and transfected with plasmids containing IL-1α or IL-1RA using Lipofectin. The supernatants were collected 48 h after transfection, and cytokines were measured by ELISA in duplicate. A, IL-1α production. B, IL-8 production. Data from one of three experiments is shown. ∗, a significant difference when compared with vector control-transfected cells.

Previously, we showed that NFκB and AP-1 are activated in and contribute to IL-8 expression in HNSCC (18, 19)
. To define the contribution of NFκB and AP-1 transcription factor activation in IL-1α-induced IL-8 production in UM-SCC-9 cells, we collected extracts from UM-SCC-9 cells that were exposed to IL-1α and incubated these extracts with oligonucleotides encoding the binding sequence of NFκB, AP-1, or control OCT-1. Fig. 3⇓
shows that IL-1α increased both NFκB (A) and AP-1 (B) binding to the oligonucleotides within 30 min, and induction was increased further at 1 h. We confirmed that NFκB and AP-1 activation could be induced by TNF-α as a positive control. Competition of binding by unlabeled oligonucleotides indicated that the DNA binding detected is specific, and the integrity of the extracts and loading was confirmed by constitutive OCT-1 binding.

Detection of constitutive and IL-1α-inducible activation of transcription factors NFκB and AP-1 in UM-SCC-9 cells by EMSA and IL-8 luciferase reporter assay. Ten μg of each whole cell extract was incubated with 32P-labeled NFκB, AP-1, or OCT-1 control oligonucleotides in the absence or presence of an excess of unlabeled competitor oligonucleotide. The mixture was then resolved on a 5% native polyacrylamide gel in 0.25 × Tris-borate EDTA with subsequent autoradiography. A, NFκB binding activity. Cells were treated by 500 pg/ml IL-1α for 30 min (Lanes 3 and 4) and 60 min (Lanes 5 and 6), or by 1000 units/ml TNF-α for 60 min (Lanes 7 and 8). Lanes 2, 4, 6, and 8, a wild-type unlabeled oligonucleotide was added in 100-fold excess for specific competition. B, AP-1 and OCT-1 binding activity. Cells were treated with 500 pg/ml IL-1α for 30 min (Lanes 2, 7, and 8) and 60 min (Lanes 4 and 9), or by 1000 units/ml TNF-α for 60 min (Lanes 5 and 10). Competition with unlabeled oligonucleotide is shown in Lanes 3 and 8. Probe containing the OCT-1 motif was used in EMSA as a control for quality and quantity of cell extract. The gels were from a representative experiment. C, IL-8 promoter luciferase reporter activity with mutation at NFκB or AP-1 sites. Cells (2 × 105) were plated in each well of six-well plates the day before transfection. The cells were transfected with IL-8 and pCMVLac plasmids (20:1) by Lipofectin for 5 h in triplicate. Promoter activation was measured by colorimetric assay by the addition of the luciferase substrate and normalized to cotransfected LacZ activity. The variability in CMV-Lac Z activity between constitutive and IL-1-stimulated samples in this and two independent experiments was 3–8%. Light bars, constitutive activity and dark bars, inducible activity after treatment by IL-1α (500 pg/ml) for 18 h. IL-1α treatment significantly induced the reporter activities in all promoter constructs (t test P < 0.05). NFκB mutation significantly reduced both constitutive and IL-1α-inducible promoter activity, whereas only the reduction in constitutive AP-1 activity by AP-1 mutation was significant (t test; P < 0.05). An experiment representative of three different assays is shown.

The role of IL-1α in the induction of NFκB- and AP-1-dependent expression of IL-8 was examined using IL-8 promoter luciferase reporter gene assays. A set of IL-8 promoter constructs with mutations introduced in the NFκB and AP-1 binding sites were used to examine the relative contribution of these transcription factors to IL-1α-induced activation. Stimulation of UM-SCC-9 cells with IL-1α for 18 h resulted in increased reporter activity of the minimal IL-8 promoter containing both the NFκB and AP-1 transcription factor binding sites (−133; Fig. 3C⇓
). The basal and IL-1α-inducible activation of IL-8 reporter activity in UM-SCC-9 was almost completely abolished with the mutation of the NFκB site (P < 0.05). Mutation in AP-1 binding sites partially decreased IL-1α-inducible IL-8 reporter activity. Although the CMV promoter in the β-Gal reporter used for determination of transfection efficiency has a putative NFκB binding site that could potentially be responsive to IL-1-induced activation, we observed a variation of <10% in CMV-β-Gal activity between constitutive and IL-1-stimulated samples in this and two independent experiments, which did not meet statistical significance.

To examine further the effects of endogenous IL-1α on NFκB activity, we tested NFκB reporter gene activity after the transfection of IL-1α and IL-1RA. A modest increase in NFκB reporter activity was observed at 72 h after transfection of IL-1α (Fig. 4)⇓
, whereas transfection of IL-1RA resulted in a significant decrease in NFκB reporter activity (Fig. 4)⇓
. Intracellular IL-1RA suppressed >80% of NFκB luciferase activity when compared with vector-transfected control (P < 0.05). These results provide evidence that endogenous IL-1α expression contributes to constitutive activation of NFκB in the UM-SCC-9 and -11B cell lines.

Effect of transfection with plasmids encoding IL-1α and icIL-1RA on NFκB luciferase reporter activity in UM-SCC-9 cells. UM-SCC-9 cells were transfected with IL-1α and icIL-1RA under the same conditions as those described in the legend to Fig. 2⇓
. NFκB luciferase reporter gene assay was performed. The results shown represent one of three independent experiments. The reduction in NFκB reporter activity upon expression of icIL-1RA was significant (P < 0.05). ∗, a significant difference when compared with control vector-transfected cells.

To examine whether endogenous IL-1α contributes to survival, UM-SCC-9 cells were cotransfected with a plasmid encoding CMV-β-Gal as an indicator for survival, along with plasmids encoding either IL-1α or intracellular IL-1RA. As shown in Fig. 5⇓
, transfecting cells with the plasmid with the IL-1α sequence resulted in increased β-Gal activity in cell cultures when compared with vector transfection. Conversely, transfecting cells with icIL-1RA significantly decreased β-Gal activity in the low IL-1α-producing UM-SCC-9 cells (Fig. 5A)⇓
and had a more modest effect upon the high IL-1α-producing UM-SCC-11B cells (Fig. 5B)⇓
. Antagonism of IL-1α resulting from icIL-1RA demonstrated a greater effect on survival of the low IL-1-producing UM-SCC-9 cells when compared with the high IL-1-producing UM-SCC-11B cell line. Because the CMV promoter in the β-Gal reporter used for the determination of activity in Fig. 5⇓
has a putative NFκB binding site that potentially also could be responsive to icIL-1 RA, it is possible that the effect seen results from the lower level of NFκB activity. To address this possibility, we conducted an additional independent experiment comparing the effect of the expression of icIL-1RA on CMV-Lac Z and RSV-LacZ activity, because the RSV promoter lacks a putative NFκB site. A reduction in RSV-LacZ as well as CMV-Lac Z reporter activity was observed, which is consistent with a decrease in the survival of cells cotransfected with icIL-1RA (data not shown).

Effect of transfection with plasmids encoding IL-1α and icIL-1RA on the survival of UM-SCC-9 cells. A, cells were cotransfected with the CMV-β-Gal reporter and with empty vector control, vector encoding IL-1α, or icIL-1RA in 4-fold excess and harvested after 72 h for measurement of β-Gal reporter gene activity, as described in the legend to Fig. 3⇓
. B, the cells were cotransfected with CMV-β-Gal reporter with empty vector control or icIL-1RA in 4-fold excess and harvested after 72 h for measurement of β-Gal reporter gene activity. ∗, a significant difference when compared with control vector-transfected cells.

Because IL-1α has been reported to stimulate the growth of human keratinocytes or transformed cervical carcinoma cells in vitro(10)
, we investigated whether exogenous addition of rIL-1α protein is able to stimulate cell growth of UM-SCC lines. Under serum-free conditions, a modest increase in cell growth of UM-SCC-9 in a 5-day MTT assay was detected with the addition of rIL-1α (250 pg/ml; Fig. 6A⇓
). UM-SCC-11B cells, which already produce ∼500 pg/ml IL-1α, grew under serum-free conditions without the addition of exogenous IL-1α, and the addition of the same concentration of IL-1α to UM-SCC-11B cells resulted in little additional increase in cell growth (Fig. 6B)⇓
. We observed no additional significant increase in growth stimulation when either cell line was treated with 500 pg/ml of IL-1α nor a decrease exceeding 10–15% after the addition of up to 500 ng/ml of anti-IL-1 antibody or rIL-1RA (data not shown). Because the limited effect of recombinant anti-IL-1 and IL-1RA on the endogenous proliferative rate was observed in the same concentration range that inhibited the IL-8 response of the UM-SCC cell lines to exogenous IL-1α, the results suggest that exogenous IL-1RA is a relatively inefficient inhibitor of the autocrine growth-promoting effects of endogenously produced IL-1α in UM-SCC cells.

Recombinant IL-1α stimulates UM-SCC-9 and 11B cell growth in MTT assay. UM-SCC-9 and -11B cells were plated at 5 × 103 cells/well in 96-well plate overnight and washed with 1× PBS three times. IL-1α (250 pg/ml) in KGM serum-free medium or KGM alone was added, and labeling reagent was added on days 1, 3, or 5 after treatment for MTT assay. The OD of cultures were measured by a microtiter ELISA reader at a wavelength of 570 nm. The results were presented from one of three independent experiments. ∗, a significant difference when compared with control vector-transfected cells.

DISCUSSION

Previously we reported that human HNSCCs constitutively express IL-1α and a repertoire of proinflammatory and proangiogenic cytokines that are potentially IL-1-inducible, including IL-6, IL-8, and GM-CSF (5, 6)
. We found that coexpression of these cytokines correlated with activation of immediate-early transcription factors NFκB/Rel A and AP-1 and demonstrated that inactivation of NFκB/Rel A inhibited the expression of this repertoire of cytokines as well as cell survival and growth (18, 19, 20)
. Because IL-1 has been shown to modulate activation of these immediate-early pathways and cytokines in response to injury, we explored the effects of modulating exogenous and endogenously expressed IL-1α on the activation of NFκB and AP-1 expression of proangiogenic cytokine IL-8 and upon the survival and proliferation of HNSCC cell lines. Herein, we provide evidence that IL-1α contributes to the expression of IL-8 through the transcriptional activation of NFκB and AP-1 and also promotes cell survival and the growth of HNSCC cell lines in vitro.

In this study, we showed that HNSCC cell lines produce functional IL-1α in their culture supernatants by demonstrating their ability to induce IL-8 production and the ability to neutralize this response by anti-IL-1α antibody (Fig. 1)⇓
. We also showed IL-1α produced by cells transfected with the IL-1α gene is functional in terms of IL-8 induction (Fig. 2⇓
and Ref. 5
). IL-1α was found to induce both NFκB and AP-1 DNA binding, which are potential activators of IL-8 in UM-SCC-9 cells (Fig. 3, A and B)⇓
. We established that mutation of the NFκB site abrogated IL-8 reporter activity, whereas mutation of AP-1 partially inhibited IL-8 reporter activity (Fig. 3C)⇓
. These results are consistent with previous evidence showing that recombinant IL-1α can activate transcription factor NFκB and AP-1 and synergistically induce IL-8 promoter activity in other cell types (23, 25)
and IL-8 production in HNSCC (5)
.

Transfection of cells with icIL-1RA significantly inhibited IL-8 production (Fig. 2)⇓
as well as NFκB activity (Fig. 4)⇓
. These findings provide evidence that endogenous IL-1α is an important inducer of constitutive activation of NFκB and expression of IL-8 in HNSCC. Constitutive activation of NFκB has been detected in other tumors that express IL-1α. In breast cancer, constitutive activation of NFκB was found in three of three estrogen receptor-negative breast cancer cell lines that produced functional IL-1α constitutively (26, 27)
. The demonstration that exogenous and endogenous IL-1α can constitutively activate NFκB and IL-8 expression in HNSCC provides evidence that IL-1 is one of the important factors involved in the transcriptional activation of proangiogenic cytokine genes by HNSCC.

The observation that UM-SCC-11B supernatant containing IL-1α is significantly more potent than a similar amount of rIL-1α suggests that other factors produced by these tumors may also contribute to the activation of transcription and to the expression of IL-8. Whereas IL-1β has similar activity, and icIL-1RA could conceivably inhibit either IL-1α- and IL-1β-induced cell activation, we have not detected IL-1β in culture supernatants in any HNSCC cell lines tested (5, 6)
. Overexpression of other factors and receptors that can potentially activate NFκB and AP-1 pathways have been detected in HNSCC, including EGF receptor (28, 29)
, the hepatocyte growth factor receptor (c-Met; Ref. 30
),6
and platelet-derived growth factor receptor (31)
. In cervical SCC, IL-1 has been shown to induce the expression of amphiregulin, a growth factor that binds the EGF receptor and promotes proliferation (10)
. Inhibition of the EGF receptor has been shown to have antiproliferative effects on HNSCC in vitro and in clinical trials, but it is not cytotoxic (29)
. It will be interesting to determine whether activation of the NFκB and AP-1 pathway by IL-1 contributes to proliferation through the expression of EGF family ligands or receptors. Alternatively, IL-1α, EGF, and other factors together may contribute to the activation of these pathways, indicating that inhibitors that target only one component of upstream activation may yield limited effects.

IL-8 induced in response to autocrine or exogenous sources of IL-1α is likely to be an important mediator of the malignant phenotype in human HNSCC. IL-8 and its CXC chemokine homologues have been shown to contribute to increased growth and metastasis of SCCs and other cancers through both autocrine and host-dependent mechanisms. IL-8 and its closely related homologues in the GRO family were shown originally to promote directly the proliferation of melanoma through an autocrine mechanism involving CXC receptor-2 (32)
. Recently we showed that the addition of rIL-8 can promote directly the proliferation of the UM-SCC-9 cells used in the present study (33)
. IL-8 and GRO chemokines have also been shown to promote chemotaxis of neutrophils and endothelial cells during angiogenesis (34)
. Treatment of mice bearing tumors with IL-8 and GRO cytokine-neutralizing antibodies have been reported to inhibit angiogenesis and the growth of lung, melanoma, and prostate xenografts in vivo(15)
. Recently we established that the murine homologue of human IL-8 and GRO-1 promotes growth, metastasis, angiogenesis, and inflammation in a homologous animal model of SCC through host responses involving CXC receptor-2 (35)
. Luca et al.(16)
demonstrated that transfection of IL-8 cDNA into human melanoma cells can also increase in vivo tumor growth and metastasis in xenograft models. Thus, proinflammatory and proangiogenic cytokines regulated by IL-1 can have important effects in the pathogenesis of cancer.

The finding that expression of IL-1 promoted and IL-1RA markedly reduced the number of transfectants that coexpress the β-Gal reporter provides evidence that IL-1 expression may contribute to the survival of the HNSCC cells. The finding that expression of IL-1RA markedly reduced NFκB promoter activity and survival is consistent with our previous data indicating that NFκB activation is important for survival (19)
. Inhibition of NFκB activation by a dominant negative mutant inhibitor-κB decreased cell survival (19)
and the resistance of SCC to cell death induced by TNF-α or radiation (36, 37)
. The cytoprotective effects of IL-1 against radiation have been reported previously and are consistent with the activation of antiapoptotic pathways (38)
. NFκB can inhibit cell death induced by TNF or cytotoxic therapies and can induce a variety of cytoprotective genes that mediate antiapoptotic signals (38, 39, 40, 41)
. Our results in human HNSCC are corroborated by a recent report by Vale et al.(42)
, who found that inhibition of IL-1 by IL-1RA resulted in decreased survival of foci of Raf- and NFκB-activated NIH 3T3 cells. Furthermore, we have obtained evidence that murine SCC cells that overexpress IL-1 and demonstrate increased activation of NFκB show increased expression of the prosurvival candidate inhibitor of apoptosis-1 (43)
. We have yet to determine directly whether the effects of IL-1 on survival of HNSCC cell lines are mediated by the antiapoptosis effects of NFκB and which NFκB-dependent genes may be involved. Development of an inducible system that will enable inducible overexpression of icIL-1RA and dominant-negative inhibitor-κB is under way to examine further the role of IL-1 and NFκB and to identify prosurvival genes regulated by these factors.

Expression of IL-RA in UM-SCC-9 was found to have potent inhibitory effects on the constitutive activation of NFκB, IL-8 expression, and cell survival, providing evidence that endogenous IL-1α expression is an important factor in the activation of prosurvival transcription factors and genes. The inhibitory effect observed with the expression of a construct of IL-1RA lacking leader sequence containing the signal for secretion was greater than that observed after the addition of high concentrations of exogenous IL-1RA (Figs. 2A⇓
, 5⇓
, and 7 and Ref. 5
). These findings suggest that IL-1 activity may not be limited to a receptor binding loop that involves extracellular secretion and suggests that IL-1α may mediate its autocrine effects in HNSCC via membrane or intracellular IL-1 receptor binding or actions that are relatively inaccessible to exogenous IL-1RA. IL-1α is produced intracellularly as IL-1α precursor, which is fully active (2)
. In normal cells, IL-1α precursor is released only during cell death and is cleaved by extracellular proteases to become mature IL-1α (2)
. In transformed cells, IL-1α can be released from cells without cell death (2)
, and we have shown that most HNSCCs secrete IL-1α spontaneously (5, 6)
. However, evidence has accumulated that is consistent with the hypothesis that IL-1α may have intracellular functions. Soluble IL-1 has been shown to bind to surface IL-1R and be internalized (44)
. IL-1α also has been detected as an intranuclear protein, where it may either directly bind to DNA or form complexes with intracellular IL-1R, which then binds to DNA (45)
. We have shown that intracellular inhibition of IL-1α activity has potent inhibitory effects on the constitutive activation of NFκB, IL-8 expression, and cell survival. Determination of the mechanisms by which IL-1α activates the transcription and expression of prosurvival genes in HNSCC and other cancers that express IL-1 may provide new approaches for anticancer therapy.

Acknowledgments

We thank Drs. Keith Brown and Ulrich Siebenlist of the National Institute of Allergy and Infectious Diseases (NIAID), NIH, Bethesda, MD for the generous gift of the plasmid IgkB-Luc and Steve Haskill, University of North Carolina, Chapel Hill, NC, for pcDNA icIL-1RA. We also thank Drs. James Battey (NIH) and James Mulshine (NIH) for their helpful suggestions.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 Supported by the National Institute on Deafness and Other Communication Disorders Intramural Research Project Z01-DC-00016 and the NIH Undergraduate Research Scholars Program (to D. E. C.).